Bovine mastitis is the most frequently occurring and costly disease affecting dairy producers. The transmittable bacterium Staphylococcus aureus is the most common cause of bovine mastitis and current antibiotic therapies usually fail to eliminate the infection from dairy herds (Sears, P. M. and K. K. McCarthy, 2003).
Multiple drug resistance (MDR), partly due to the excessive and improper use of antibiotics both in human medicine and food animal production, is a growing problem that has come to the forefront particularly in the last decade. A major cause of this widespread multi-drug resistance is the fact that recent drug design has been largely based on a limited number of new chemical scaffolds, allowing pathogens to adapt and circumvent common antibiotic action mechanisms (Blount and Breaker, 2006). Staphylococcus aureus and Clostridium difficile, which are particularly prone to developing antibiotic resistance, are nosocomial pathogens of major importance responsible for a significant mortality rate in hospitals and increased health care costs (Talbot et al, 2006). Alternative antibacterial drugs targeting RNA, mainly based on a fortuitous interaction between an exogenous ligand and its RNA target, have recently been developed and may help alleviate the problem of MDR (Knowles et al, 2002). However, there remains a great need for the identification of novel antimicrobial targets and compounds.
Bacterial riboswitches as a mechanism for regulating gene expression are known in the art. Riboswitches are segments of the 5′-untranslated region of certain mRNA molecules that, upon recognition of specific ligands, modify the expression of one or more proteins encoded in the message. Ligand-binding results in structural changes in the riboswitch that affect the ability of the mRNA molecule to be properly transcribed or translated. Thus riboswitches can function as RNA sensors, allowing control of gene expression at the mRNA level. The best-characterized riboswitches contain an aptamer region that is involved in ligand binding and an expression platform that is responsible for bringing about changes in gene expression (Coppins et al., 2007).
The scientific literature suggests that all bacterial riboswitches indiscriminately represent molecular antimicrobial targets. However, in order to develop novel specific antimicrobial therapies that will avoid contributing to widespread MDR, it would be highly desirable to identify riboswitches that are therapeutically selective, i.e., riboswitches that do not represent antimicrobial targets in all microbial species or commensal microorganisms that bear similar switches but on the contrary, riboswitches that have specific properties that only appear in the targeted pathogens.
Guanine riboswitches represent a subclass of riboswitches that are known in the art (Barrick and Breaker, 2007; Batey et al, 2004; Mulhbacher and Lafontaine, 2007). Application WO 2006/55351 teaches that the guanine riboswitch, in the presence of the guanine ligand, inhibits the expression of the genes xpt and pbuX in the non-pathogenic bacteria Bacillus subtilis. Some of the guanine analogs proposed in application WO 2006/55351 as potential therapeutic agents were reported to have non-specific antibacterial activities in vitro against a broad selection of pathogenic and non-pathogenic bacteria including the non-pathogenic Bacillus subtilis. Furthermore, later studies from the same investigators reported that these two genes (xpt and pbuX), assumed to be controlled by the guanine riboswitch, were expected to be non essential for the survival or virulence of Staphylococcus aureus (Blount and Breaker, 2006). It is well known in the field that genes (and their products) that are essential for the survival or virulence of a microbial pathogen represent ideal targets to obtain quick microbial killing, while avoiding the development of microbial resistance arising from mutations inactivating non-essential anti-microbial targets. The teachings of WO 2006/55351 and of Blount and Breaker (2006) thus suggested that the guanine riboswitch was not a desirable target for selective antibacterial intervention against the pathogen S. aureus in vivo.
Hence in this field of research and therapeutic applications, it would be highly desirable to identify microbial pathogens having essential survival or virulence gene(s) under control of a riboswitch. It would also be highly desirable to identify antimicrobial compounds that specifically bind to these riboswitches and selectively inhibit the expression of the gene(s) essential for the survival or virulence of specific pathogens in vivo. Such compounds would allow selective treatment of pathogens affecting animals and humans. It would also be highly desirable to identify compounds that are sufficiently distinct from the natural riboswitch ligand to avoid transformation of the compound by the host or microbial flora (for example by ribosylation) and to prevent potential broad and non-specific bacterial growth inhibition or toxicity to the mammalian host.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.